Endometriosis: Not just a gynecologic Disease - Hugh Taylor, MD

I am Hugh Taylor from the Yale School of Medicine, and it's a pleasure to be here remotely with you today. I wish I could be there in person to see all of my friends, but today I'm going to talk to you about endometriosis. And what I want to stress today is that endometriosis we think of as a gynecologic disease, but there are many systemic manifestations of endometriosis. This is really not just a gynecologic disease. We've traditionally defined endometriosis as ectopic endometrial glands and stroma that is in the pelvis, and we see multiple types of lesions. That simple histologic definition of endometriosis really belies the complexity of this disease. Sometimes we see the blue or brown lesions, the powder burn lesions. Sometimes we see red lesions, white lesions, clear lesions are the big endometriomas. This is a complex disease that is more than that simple histologic definition would imply.

Not only that we know the clinical presentation is quite varied. Sometimes we see patients who present with horrific pain. Sometimes they have no pain at all, and we find that we diagnose it when we see them for infertility treatment. Sometimes they have gotten pregnant, have no pain, and we find it completely asymptomatic. It's seen as a incidental finding on an imaging technique for some other purpose or a surgery for some other reason. So the clinical presentation is quite varied and doesn't point to this just being simply a localized disease to the pelvis. Further, we know that the pain varies considerably or rather, doesn't vary by stage of the disease that whether we look at dysmenorrhea or non menstrual pelvic pain or dyspareunia at any stage of the disease, we're just as likely to see pain. So what's going on? What we see in the pelvis doesn't explain the entirety of this disease.

And then finally, endometriosis is associated with many other symptoms outside of the pelvis. We see, of course the infertility and pain that are the classic things we think of, but we know there can be bladder dysfunction, there can be whole body inflammation, bowel dysfunction. We know that anxiety is more common in women with endometriosis, that they can have a lower BMI, that they can have depression fatigue, and that in the long run there even now, we know more likely to develop cardiovascular disease. So what I want to stress is what you see in the pelvis is really just a part of this disease. Endometriosis truly is a systemic disease that affects the entire body and multiple organ systems. And referenced here as a review we wrote a few years ago that really summarizes much of this concept of all the different organs that are affected.

Well, we think of traditionally of endometriosis as being due to retrograde menstruation and s samson's theory, but this absolutely cannot explain all of endometriosis. It cannot explain the systemic effects. It can't explain endometriosis outside of the peritoneal cavity and it can't explain those rare cases where we find endometriosis. For example, in men in the old days when we treated men with high doses of estrogen for prostate cancer, some would develop endometriosis. There have to be other etiologies of endometriosis. We've championed the stem cell theory can stem cells from bone marrow and other sources differentiate into endometrium and other locations. To summarize this data from many years ago now, about 20 years ago, we first started looking at mesenchymal stem cells in the bone marrow. These multipotent stem cells in the bone marrow that could differentiate into multiple cell types in solid organs. And we reason certainly they should be able to give rise to endometrium one of the cell types with the most dramatic turnover.

Well, we looked first in a mouse model. We took cells from male mice and bone marrow derived cells and we transplanted them into females. And then we looked at Y chromosome by fluorescent insight to hybridization, and this is what we saw. The A, it shows you a control male. The blue is the nuclei, the red dots of the Y chromosome, and you can see that almost every cell we can see that Y chromosome B is a female to female bone marrow transplant. Looking at the uterus, there are none. But in C and D you can see those Y chromosome cells that came from the bone marrow that we transplanted into these mice in the uterus. The C shows the epithelium and the dark stripe. Down the middle is the lumen of this uterus. These are the epithelial cells on either side and that's a bone marrow derived cell.

And D is the stromal cells. We also looked in humans and we looked at women who'd had bone marrow transplant in the old days when we took whole bone marrow, transplanted it in in. And here we looked at a woman who had a single HLA mismatch in her bone marrow donor. And the brown hair stands for the HLA type of the bone marrow donor. So you can see on the left the bone marrow cells that interpolate into this endometrium. The epithelial cells that are bone marrow donor origin on the right, the arrows point to the stromal cells that are bone marrow donor origin. The arrowhead point to the cells that are the endogenous resonant stromal cells that don't have that HLA mismatch. Those are the cells that have the same HLA type as the recipient. And of course we looked at multiple other markers to prove that these cells really are endometrial cells that have differentiated from bone marrow and not just leukocytes that are migrating there.

We also show that they contribute to the ectopic lesions. This is a busy slide, but I want to show you to the right. You can see that those red cells or cells coming in from bone marrow, this is a bone marrow transplant with a cells that had red fluorescent protein. You can see not only do they contribute to the endometriosis lesion itself, but outside of that endometriosis lesion, they help propagate it elsewhere and contribute to the blood vessels that come in and support that lesion. The angiogenesis. So this is a novel origin for endometriosis. Stem cells contribute to endometriosis. They likely account for endometriosis outside of the peritoneal cavity. And truly a novel mechanism, not just endometriosis, but any disease. And I imagine we'll find some other diseases are due to stem cell inappropriate ectopic differentiation. But not only that, these stem cells from the endometriosis that go there, some of them remain a stem cells, they're altered, and then they can come back into the circulation.

And this is a mouse experiment. We see these peaks of endometrial or endometriosis derived stem cells that travel then in the circulation and they can go to other organs. Is this a way that so many different organs are affected not only by endometriosis, either coming from bone marrow derived stem cells or from the endometriosis lesions themselves and then going to distant organs where they may have an effect? And this is some cell sorting experiments we did. We created endometriosis lesions with red fluorescent protein. You can see on the left the panel there looks at four different organs, lung, spleen, liver and brain. And the left shows the host organs from which we derive the endometrial cells for the transplant. The middle column shows the negative control where we do not use the red fluorescent animals. And then the third column shows the endometriosis created from those animals with the red fluorescent protein.

And we look at those four different organs and to the right of that line, you'll see a few cells in each of these that have that red fluorescent protein in the lung, in the spleen, in the liver and the brain. They're rare, small numbers, nothing you would ever clinically recognize as endometriosis, but could they be altering the function of these different organs in women with endometriosis? And again, this looks at the percent cell count. You can see it's tiny. But in every mouse we looked at and every organ we looked at here show the main four we looked at. You can see some of these DS red cells or these red fluorescent cells in these organs. So when we think about cell trafficking and endometriosis, this really is a disease of cells moving around the body. We've always thought of it that way from the old theories of retrograde menstruation where cells come out of the uterus and traffic to the peritoneal cavity.

But now we know that cells from bone marrow can go both to the uterus and feed the endometriosis, and I draw that line much thicker going to the endometriosis because that inflammatory environment in the endometriosis attracts more stem cells and then those cells can leave the endometriosis and contribute to or influence a host of other organs throughout the body. Could this be a way where we see the mechanism that mediates those non pelvic manifestations of endometriosis? The other mechanism we looked at were microRNAs. These are small RNA molecules that are in the genome, but they don't encode proteins. They're highly processed into small, approximately 22 nucleotide RNA molecules that in general bind messenger RNA and block its transcription rather it's translation. We've shown some time ago that microRNAs, this looks at the seven family of microRNAs are decreased in endometriosis and in the more aggressive endometriosis, the red bars are essentially DEI at even lower levels of let seven.

What does let seven do? Well, I mentioned to you that let seven is involved in blocking the translation of messenger RNAs, including those of cyclones, cycl dependent kinases or kras M others genes involved in mitotic signaling genes involved in angiogenesis, cell adhesion, cell migration, all the things that go wrong in endometriosis generally let seven would block these processes. And remember I said let seven is lower in endometriosis, so all of these processes go unchecked. Well, these microRNAs not only have that local effect in the endometriosis, but we know that they're either secreted alone or in exosomes where they can travel in the circulation to distant organs. We looked at these as potentially even serving as biomarkers for endometriosis. And indeed we showed that there's some microRNAs that are increased in endometriosis, some that are decreased. And we actually showed that you can make a pretty good diagnostic test for endometriosis using these microRNAs with a very high area under the curve of this receiver operator and characteristic curve here.

So potentially can even use microRNAs as a diagnostic, but that's not what I'm here to talk about today. I'm talking about the whole body effects of endometriosis. So when we know from tumor biology and cancers, when these exosomes go into circulation, they can reach shells far away from where they're made and have a hormone like effect influence gene expression, block RNA translation in cells very far from where they're made. So we looked to see if that could be going on in endometriosis. First we looked at immune cells. This is macrophages in women and we've treated them with the micro microRNA compliments that would mimic what was happening in endometriosis. The two black bars show mimicking endometriosis and the gray bars of control. So when we either increase microRNA 1 25, which is increased in endometriosis, or we use an inhibitor to let seven B, which is decrease in endometriosis, we get an increased expression of TNF, alpha IL one, beta IL six IL eight.

These circulating microRNAs are affecting macrophages throughout the body to increase the production of these inflammatory cytokines that have been associated with endometriosis. So these microRNAs are in the circulation. They're going to affect every macrophage throughout the body. And when we correlate the levels of these inflammatory cytokines, the amount of microRNA 1 25 in the circulation, we can see it as highly correlated in the endometriosis patients that at least for TNFL file one beta and IL six, a very close correlation between microRNA 1 25 and these inflammatory cytokines. The other thing we know is there's a metabolic phenotype in endometriosis where we know women with endometriosis tend to have a lower BMI and a lower fat content. Well, we showed in animal models that this is cause and effect. We create endometriosis in the animal models and the mice don't gain as much weight, so it's directly caused by the endometriosis.

You'll still see in some textbooks where it says that low body weight is a risk factor for endometriosis. No, this is caused by the endometriosis. How does this metabolic effect happen? Well, I won't go through all the details in this slide, but we looked at the liver of animals after we created endometriosis, and you can see changes in a lot of the genes that would be predicted to lead to a lower body weight. So the metabolic effect on the liver is part of what produces this. We also looked at adipose tissue here. We directly treated with microRNAs to mimic what we see in endometriosis. Different microRNAs here, 3 42 is one of the main players, but it changes leptin, adiponectin IL six, hormone sensitive lipase in this adipose tissue contributing to this metabolic phenotype we see in endometriosis. So these microRNAs go to the liver, go to the adipose tissue and change metabolism.

Other thing we looked at was brain. Now we know women with endometriosis can have central sensitization of pain. We know they can have increase in anxiety and depression, and we show here an animal models where we can really get it cause and effect that these are not just simply patients that are more sensitive to pain or patients that are anxious and complaining more. This is really a direct effect. These behaviors of creating endometriosis. Here we show when we create endometriosis in our animal model and the red bars of the endometriosis, we take out the brains and we do immunohistochemistry. Various genes that are associated with these phenomenon are altered in the brains of animals with endometriosis. On the upper right, we show the electrophysiology is different, but most importantly, when we look at behaviors, we show these behaviors are changed by creating endometriosis. You can see anxiety.

We measure this in the open field test where we see how much time the mice huddle in the corners. That tends to be a sign of anxiety in mice, and you can see that within about six weeks we see a statistically significant decrease in the amount of time they stent in the center of the box. They spent more time in the periphery of the box, again induced by the endometriosis. When we look at depression here in the bottom right, you can see here that depression was increased within the first week of creating endometriosis. And when we look at pain sensitization here, we use a hot play test where we put the mouse's paw on a warm surface, see how long it takes for that mouse to remove the paw, sensing that that's hot, and you can see that it removes it much faster. In my with endometriosis, it took about 12 weeks for that to become statistically significant, but it was so all of these manifestations are due to the effect of endometriosis on the brain.

We induce anxiety, pain, sensitization, depression, all from creating endometriosis. And then we looked at atherosclerosis. We took a APOE mouse because mice just generally don't develop atherosclerosis like we do. We took an APOE mouse and we created endometriosis. And you can see on the left the oil red o staining, it stains for atherosclerosis in this aorta of this mouse. It's much higher in the mice with endometriosis. And when we look at the luminal opening, the size of the opening of the blood vessel, it's decreased in mice with endometriosis. And if we look at the thickness of the aortic wall, again, suggestive endometriosis, much thicker in mice with endometriosis, endometriosis is causing this atherosclerotic effect that is not due to medications we give our patients. It is not due to our surgeries on our patients. It's a direct effect of the endometriosis. What mediates this? Well, we didn't see any changes in lipid levels when we created endometriosis, but inflammatory markers were quite increased as was vegf.

So we believe that's what's driving that atherosclerosis. So I hope I've convinced you that endometriosis really is a systemic disease that we're just seeing the tip of the iceberg and what we see in the pelvis that these many of these different effects of microRNAs, of stem cells, of inflammatory cytokines all drive change in other organs, but endometriosis really needs to be considered a chronic and systemic disease. The very presentation really have made it difficult sometimes for our colleagues. Probably not anyone in this group was an endometriosis specialist, but for some practitioners to diagnose endometriosis, we know it takes seven to 12 years from the onset of symptoms till we typically make the diagnosis. Many primary care doctors aren't familiar with the disease, but these symptoms that are beyond the pelvis are nonspecific, associated with all sorts of other disorders, so really can make it confusing.

I think this is one of the major barriers we need to start thinking about people with pelvic pain and depression as not needing a psychiatrist. People with bowel pain, with bladder pain, with fatigue as all manifestations of the endometriosis and not think about multiple diagnosis. And I'm sure you've all had patients who come to you after seeing the urologist or the gastroenterologist or even the psychiatrist when they really just had endometriosis. I think we know, we see that endometriosis is misdiagnosed in the majority of the cases, and it takes half of women over five physicians before they get the right diagnosis. We can do better, and part of it's going to be understanding the systemic nature of the disease. It is a widespread systemic disease. The systemic nature explains these symptoms often associated with endometriosis. We need to start thinking about this as one disease. Recognize all of these as just simply manifestations of endometriosis and the mechanisms include stem cells, microRNAs, inflammation, all these circulating inflammatory cytokines. These are the mechanisms that mediate these long range effects of endometriosis. I'll end there and acknowledge all of my collaborators at our endometriosis center, my laboratory, me members and funders that are listed here. Thank you.